【作者】 张晓；

【导师】 于源华；

【作者基本信息】 长春理工大学 ， 无机化学， 2007， 硕士

【摘要】 本研究旨在探索HBV S／HEV ORF2融合基因在植物中表达乙肝戊肝双价抗原的可能性，以期为进一步研制植物乙肝戊肝双价口服疫苗打下基础。 应用重组PCR技术将乙肝病毒（HBV）S基因连接在戊肝病毒（HEV）ORF2部分序列的5′端，二者通过柔性肽（Gly₄Ser）₂序列相连，构建成融合基因BE，测序结果正确。 将融合基因BE克隆到植物双元表达载体pBin438上，获得pBin438BE，然后用冻融法将其转入根癌农杆菌EHA105中，侵染丽春番茄子叶和下胚轴，经预培养、共培养、筛选培养、生根培养获得了5株抗卡那霉素的番茄植株。提取抗性植株基因组DNA，进行PCR及PCR-Southern检测，4株获得阳性信号。初步表明目的基因已整合到了番茄基因组中。更多还原

【Abstract】 This research was to explore the feasibility of expressing HBV S/HEV ORF2 fusion gene in plants for the purpose of developing a new type of plant-derived hepatitis bivalent edible vaccine.Hepatitis B Virus S gene was connected to the 5’ end of Hepatitis E Virus ORF2 partial sequence with the technique of recombinant PCR,a designed DNA sequence which encoded a polypeptide linker （Gly₄Ser）₂ was used to splice the two fragments. DNA sequencing results showed that the fusion gene（BE） was successfully constructed.The fusion gene（BE）,approximatelyl300bp,was cloned to the plant binary expression vector pBin438.The recombinant plasmid pBin438BE under the control of DE35S promoter was constructed and transformed into Agrobactrium tumefaciens EHA105 via freeze-thaw method. Inoculated explants from cotyledons and hypocotyls were cultured successively on pre-culture medium co-cultivation medium selective medium and root-growth media 5 Km-resistant tomato plants were obtained .The regenerated plantlets were analyzed further by PCR and PCR-Southern blot.The results confirmed that the fusion gene had been integrated into tomato genome.更多还原